Fig. 6.

USP35 isoform 1 is an anti-apoptotic protein. (A) Expression of the indicated isoforms of USP35 in stably transfected U2OS FlpIn cell lines was carried out for 48 h. Cells were lysed, and samples were resolved by SDS-PAGE and immunoblotted (IB) with antibodies against the indicated proteins. cl. BAP31, cleaved BAP31. (B) HEK293 FlpIn stably transfected cell lines were induced to express FLAG-tagged USP35^iso1 wild-type (WT) protein, its catalytically inactive variant (C450A) or empty vector control. At 24 h post induction, cells were treated with 50 ng/ml TRAIL. Cells were lysed, samples resolved by SDS-PAGE and processing of caspase-8 analysed by immunoblotting. (C) HEK293 FlpIn parental cell line and two USP35 knockout clones (denoted 7-6 and 7-10) were treated with 50 ng/ml TRAIL and samples were analysed as for B. (D) As in C, but the cell lines were seeded into a 96-well plate, incubated with 50 ng/ml TRAIL for the indicated times and MTS proliferation assay was performed. The values on the y-axis represent the ratio of absorbance read for treated to untreated cells. Error bars indicate the s.e.m. for n=3 biological replicates. (E) HEK293 FlpIn parental cell line and the two USP35 knockout clones were treated with increasing concentrations of staurosporine for 24 h. Cells were lysed, and samples analysed by immunoblotting with an antibody against cleaved caspase-3. (F) HeLa cells were transfected with wild-type USP35^iso1 or a variant with Asp743 replaced with an alanine residue, both tagged at the C-terminus with a GFP tag. At 24 h post transfection, 1 µM staurosporine was added and cells incubated for the indicated time. Cells were lysed, and samples were resolved by SDS-PAGE and analysed by immunoblotting against the indicated proteins.